Method for production and use of pathogenic fungal preparation for pest control

ABSTRACT

The present invention provides a process, formulations, and method of using novel biopesticides comprised of a prilled formulation comprising a carrier and a pathogenic fungal mycelium.

This application is a continuation-in-part application of U.S. Ser. No.772,983, filed Oct. 7, 1991, abandoned which is a continuation-in-partof U.S. Ser. No. 639,641, filed Jan. 10, 1991, abandoned.

TECHNICAL FIELD

The present invention relates to novel methods and fungal compositionsfor the biocontrol of pests. More specifically, the present inventionrelates to novel entomogenic fungi biopesticidal compositions, themethod of producing the same, and the method of using the same for thecontrol of pests. The biopesticides are particularly useful incontrolling pests which cause significant amount of damage to plants(e.g. soilborne pests and plant pests). A preferred entomogenic fungususeful in the present invention is Paecilomyces fumosoroseus ATCC 20874.

The invention also provides novel methods for preparing and preservingfungal biopesticidal materials, and methods of applying and using suchbiopesticidal formulations to obtain maximum efficiency in controllingvarious soilborne pests, plant pests, mosquitoes and soil nematodes.Methods for effective quality control of biopestices disclosed hereinare also provided.

BACKGROUND

Chemical pesticides have been used intensively to control pests for manyyears. An awareness of recent problems in the use of pesticides andconcern about their adverse effects on man and his environment haveresulted in more commercial attention being given to biological controlalternatives. Certain entomogenic fungi have been recognized byresearchers to be pathogenic to different pests; particularly the use ofentomogenic fungi has been widely studied as biological control agentsin the USSR and in Europe. Update reviews on the different pathogenicfungi and their use and status can be found in: Carlo M. Ignoffo and B.Handava "Handbook of Natural Pesticides", vol. V, part A., C. W. McCoyet al. "Microbial Insecticide", CRC Press, 1988, and M. N. Burge "Fungiin Biological Control Systems", Manchester University Press, 1988.Unlike insect pathogenic bacteria or other microorganisms (e.g., virusesor protozoa) which must be ingested by the insect to initiate diseases,entomogenic fungi normally invade through the host's cuticle.

Entomogenic fungi infect pests, usually insects, by a parasitism diseasemechanism. The infection process development is believed to consist ofthe following steps:

1. Attachment--The conidium of the entomogenic fungi spore is attachedto the insect cuticle.

2. Germination--The conidia spore is germinated on the insect cuticle toform a germ tube.

3. Penetration--The germ tube penetrates directly into the cuticle. Itis believed that the cuticular invasion involves both enzymatic andphysical activities.

4. Growth--The fungus grows in the hemocoel as mycelium or blastospore.The fungi overcome the host by invasion of organs.

5. Saprophytic Growth--The fungi grows on the outside of the insect andproduces aerial conidia spores.

Some entomogenic fungi overcome their host before extensive invasion oforgans takes place, presumably by production of toxins. Although toxiccompounds have been reported from culture filtrate of mycelium ofseveral entomogenic fungi (e.g., Paecilomyces fumosoroseus was shown toproduce the toxin Beauvericin (Ferron, Annual Review of Entomology23:409-442 (1978), and a peptide toxin known as destruxin A has beenisolated from culture broth of Metarrhizium anisoplia (Y. Kodaira, Agr.Biol. Chem. Chem. 26-36 (1962)), only in few cases it was reported thattoxins have been detected in insects infected with the entomogenic fungi(Suzuki et al., Agric. Biol. Chem. 35:1641-1643 (1971)).

The use of the entomogenic fungi for control of different pests is notitself a new idea. Entomogenic fungi such as Metarrhizium, Beauveria,Hirsutella, Verticillium or Paecilomyces have been studied fordevelopment as pest control agents. Solid state fermentation has beenwidely examined because this method allows the production of theinfectious bodies of the entomogenic fungi, e.g., conidia spores.However, it has been found that conidia spores of the variousentomogenic fungi are very sensitive to drying processes and the conidiaspores lose their viability very quickly.

Submerged fermentation have significant problems to overcome. Most ofthe entomogenic fungi heretofore grown in submerged culture producemostly blastospores with some mycelium. Blastospores are not stableduring storage or drying processes. Attempts to formulate blastosporesresulted in reduced efficacy and stability.

In order to overcome the above stability problems associated withconidia spores and blastospore preparations, several processes have beendisclosed in the literature for the production of biocontrol agentsbased on biomass obtained from submerged culture fermentation. Thesetechniques generally involve growing fungi in liquid media followed byinoculation of a solid media or an inert carrier, such as vermiculite,on which the conidia spores are produced. For example, Kybal (1976)discloses a process for incubating biomass containing mycelium,blastospores and other fungal stages in shallow, aerated vessels toproduce conidia spores directly on the surface of the coated vesselsurface. However, this process is very labor intensive and the conidiaspores obtained is only on the vessel surface. The surface area perapparent volume tends to be small and the efficiency is low.

In another process McCabe et al., (U.S. Pat. No. 4,530,834), disclosesthe use of entomophthoralean mycelium produced in submerged culture forthe production of resting conidia spores. This process is a diphasicsystem whereby production of spores or blastospores is bypassed. First,the biomass produced by liquid fermentation is dried with protectiveagents, and the dry biomass mat obtained is milled to a dry powder form.Next, the dry powder preparation is rewetted and applied to the targetpest. However, this method is plagued with stability problems. Thepowder product needs to be stored below 4° C. to maintain its viability.Even under such storage conditions the product is only stable for 64days. Further, conidia spores produced from reconstituted powder areshort lived and fragile.

Dried mycelium particles obtained from liquid fermentation of differententomogenic fungi have been produced in similar ways by differentinvestigators. For example, McDonald et al. (Vth InternationalColloquium on Invertebrate Pathology and Microbial Control, Adelaide,Aus, p. 147) disclose a similar basic process for the entomogenic fungusCulicinomyces clavisporus for mosquito control. This process involvesgrowing the fungus in liquid culture for 6 days, harvesting thesuspension by filtration and adding sucrose followed by air drying. Thedried mycelial mat was then ground in a hammer mill and sieved through a3.5 μm sieve. Upon addition of these mycelium particles to waterapproximately 5×10⁶ conidia spore/mg of dried particle were produced.Dried mycelium particles could be stored for 1-5 weeks at 4° C. withoutlosing activity. A similar approach has been used by Roberts et al. (VthInternational Colloquium on Invertebrate Pathology and MicrobialControl, Adelaide, Aus, p. 336) for the use of dried mycelium particlesof Metarrhizium anisopliae isolate ARSEF 2457 to control the Japanesebeetle and other pests. Based on the Roberts et al. results it has beenfound that lyophilized Metarrhizium mycelium has a longer shelf life insoil than conidia spores.

Bayer A. G. (EP application 0268117 A2, 1987) has reported on a similarmethod which is based on the production of mycelium and blastospores ofMetarrhizium anisopliae in a fermenter. During the course offermentation, the mycelium/blastospore aggregate to form pellets in thesize of 0.1 mm up to 1.5 mm in diameter. At the end of the submergedculture fermentation, the mycelium/blastospore aggregates are harvestedand dried in a fluidized bed dryer to form a final product whichconsists of dry mycelium/blastospore granules with 0.5 to 1.5 mmdiameters. The granules can be applied to soil where they can forminfectious conidia spores. However, this method suffers from severalproblems. First, the fungal pellets formed in the fermenter result in avery low biomass yield. Therefore, such a fermentation process is noteconomical. Also, the final granular product obtained must be storedunder vacuum and at a low temperature. It seems also that the conidiaspores formed after activation in the soil have a short life. This isprobably because the granules do not contain any nutrients which canstimulate or promote growth.

Another approach, which has been used widely for the delivery ofdifferent fungal pathogens (mainly conidia spores and chlamydospores)against plant diseases, is encapsulating fungal spores in alginateprills (Lewis et al., Proceed. Intern. Symp. Control. Recs. Bioact.Mater, 12:341-3 (1985), Fravel et al., Phytopathology, 27:3341-8 (1982),and J. J. Morois, U.S. Pat. No. 4,724,147). This method has been ignoredin general for the delivery of entomogenic fungi for insect and otherpest control. Few publications on the use of alginate prill for deliveryof entomogenic fungi are known. For example, Enrique A. Cabanillar(Ph.D. thesis) "Factors Influencing the Efficacy of Paecilomyceslilacinus in Biocontrol", 1987, North Carolina State University,described this method to deliver conidia spores of Pacilomyces lilacinusproduced on solid media (solid state fermentation) against Koot-knotnematode. Roberts, et al. (Entomopathogenic Fungi: Recent Basic &Applied Research; Matha, V. et al. (ed) Biopesticides Theory & PracticeProc. Conf., Sep. 25-28, 1989, Ceske, Budejovice, Czechoslovachia 11-30pp) disclose a process for using alginate pellets which incorporate themycelium biomass of Beaveria bassiana & Metarrhizium anisopliae withpre-gelatinized starch as a basic nitrogen supply.

SUMMARY OF THE INVENTION

This invention provides a process and formulation for the production ofbiopesticides comprising mycelium and conidia spores of entomogenicfungi. The process offers a new opportunity to use entomogenic fungi inthe control of soil pests, plant pests, nematodes, and mosquitos. Themethod of the invention is particularly useful to prepare pesticidialcompositions comprising the entomogenic fungus Paecilomyces fumosoroseusATCC 20874 (U.S. Pat. No. 4,942,030). However, a variety of fungi havingpesticidal properties can be used in the process and formulations of theinvention to control various pests.

This invention provides a process for preparing a biopesticide usefulfor controlling or preventing pest infestation. The process includesfermenting one or more species of fungus effective for control of pestsin a culture medium by submerged fermentation to produce biomass suchthat at least about 80%, preferably 90% and most preferably 95% of thebiomass is in the form of mycelium. The mycelium is then harvested andmixed with a carrier. The biomass/carrier mixture is formed into prilland dried. The prill can be either used directly as a dry formulation orthe prill can be used as a carrier for sporulation of conidia spores(reactivated prill) or the harvested conidia spores can be used or theharvested conidia spores can be pregerminated and then used.

Generally, this method comprises the production of mycelium prills ofthe entomogenic fungi. The process for forming prills is divided intothree main stages as follows:

Stage I: Production of fungal biomass in very high yield in fermenter inthe form of filamentous mycelium.

Stage II: Harvesting the filamentous mycelium, formulating the wetmycelium, and encapsulating the formulated wet mycelium in calciumalginate prills.

Stage III: Drying the formulated prills and packaging the product foruse as a biopesticidal agent.

The method can further comprise the following stages:

Stage IV: Activating and cultivating the prill to produce pathogenicconidia spores.

Stage V: Drying the reactivated prill and packaging the product for useas a biopesticidal agent.

It is within the scope of the invention to dry the prill before or afteractivating and culturing the formulated prill.

The method can further comprise the following stage:

Stage VI: Harvesting the pathogenic conidia spores from the reactivatedprill and using the conidia spores as a biopesticidal agent.

The method can further comprise the following stage:

Stage VII: Pregermination of the pathogenic conidia spores harvestedfrom the reactivated prill and using the pregerminated conidia spores asa biopesticidal agent.

For purposes of this invention, the term "biopesticide" or"biopesticidal agent" shall mean a biologically-active pathogenic fungalagent which is useful in the control or prevention of plant-, soil- orwater-borne pest infestation by adversely affecting the existence orgrowth of the target pest, particularly insects. Such control cancomprise a complete killing action, eradication, arresting in growth,reduction in number, induction of plant resistance or the production ofphytoalexins or any combination of these actions. The term "entomogenic"as employed in the specification and claims of this invention shall meanpathogenic to insects specifically but can also be construed broadly tomean pest pathogen. The term "control" or "biocontrol" as employed inthe specification and claims of this invention is to be construed asmeaning protecting plants, soil or water from pest, particularly insect,damage by use of the biopesticides of this invention. The term"pesticidally effective amount" or "effective amount" is used herein tomean the amount of biopesticide sufficient to control pests, inparticular insects.

As described in the background, at present there is no effective stabledelivery system available for entomogenic pesticides.

A primary object of this invention is to provide a process for producingan economical entomogenic fungal biopesticide product. The processproduces a fungal biocontrol material that is easy to handle and toapply in either horticultural or agricultural settings. The dried fungalproduct prepared as described herein is easily produced, stored,shipped, and formulated to control plant, soil, and water pests. Theproduct can be stored at room temperature, preferably between 10° to 25°C., for extended periods, i.e., more than a year, without losingconidiation activity and prill/spore viability.

Still another object of this invention is to provide a process ofconverting an actively growing culture of an entomogenic fungus into aformulated biopesticide which is easily handled and applied. Theformulated prill of the invention maintains biological activity of thefungal product until the time of application. Without the prillformulation, the mycelium could not survive under such conditions. Uponapplication to to a desired locus and upon rewetting, the dried prill is"activated", or reconverted into a biologically active form, in whichmycelium budding from the prill occurs in less than 48 hours afterwetting. As the mycelium budding begins consuming the nutrients providedby the prill formulation, conidiation begins, resulting in theproduction of conidia spores. Conidia spores are the biologically activeform of the fungus which is pathogenic to pests.

Another object of this invention is to provide a reactivated carrierwhich is suitable for efficiently sporulating a culture of theentomogenic fungus to produce conidia spores in high concentrations. Theconidia spores produced from the reactivated carrier have high viabilityand infectivity and are produced in higher concentrations thanpreviously known methods. Upon harvest, the conidia spores can beapplied directly to the plant, soil, seed or root with or withoutformulation material. Either reactivated or non-activated carrier, i.e.,prill, can be applied to soil. Further, the conidia spores harvestedfrom activated prill can be transferred into a aqueous suspension andapplied to the locus of the plant. For purposes of this invention, theterm "locus" is used to describe the location wherein treatment isdesired. The locus may be in or on the surface of the soil, on the plantitself or on the seed or root thereof, or on the surface of water.

Another object is to provide a method to pregerminate the active conidiaspores harvested from the prill and apply the pregerminated sporesdirectly to the plant locus with or without formulation material.

Another object of this invention is to provide a biopesticideformulation/carrier that is useful in the prevention or control of pestinfestation, including, but not limited to, plant and soil-borne insectsand nematodes and water pests, such as mosquitos. An additionalobjective of the present invention is to provide an alternative tochemical pesticides.

It is a further object of this invention to provide a method forutilizing as a biopesticide a selected strain of the entomogenic fungal,species Paecilomyces fumosoroseus ATCC 20874 (PFR) which has a highlevel of infectivity for different plant pests. It is still a furtherobject of this invention to provide a biopesticide containing a selectedstrain of the fungal species PFR which can be readily mass produced asrequired for horticultural and agricultural applications.

Another object of this invention is to provide a biopesticidalpreparation comprising a fungal mycelium preparation of Paecilomycesfumosoroseus which is produced in a submerged culture. The mycelium iscombined with a carrier. The mycleium/carrier mixture is then prilled toprovide high quality control, stability, shelf life, infectivity, andspecificity in infecting various plant pests.

Another object of the invention is to provide an economical method forefficiently culturing fungal spores for field or greenhouse application.The method of the invention enables the production and use of largevolumes of biopesticide in an easy and convenient manner.

Still another object of this invention is to provide a novel method forproviding pathogenic fungal conidia spore biopesticide having improvedstability, viability any pathogenicity over longer periods of time thanconidia spore or fungal matter produced by previously known methods. Themethod provides major economical and commercial advantages in thestorage, delivery and application of such a biopesticide.

It is further an object of the invention to provide a method of storingand delivering the sporulated reactivated biopesticides of the inventionby placing the reactivated prill having conidia spores attached in awater-soluble polymeric container. Pesticidal formulations of theinvention are prepared by dissolving the polymeric container plusbiopesticide in an aqueous solution. A novel composition ofwater-soluble polymeric container and reactivated prill, and the methodof use thereof are to prepare novel biopesticidal formations are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the biological control of adult whiteflies usingformulation of the invention vs. chemical treatments.

FIG. 2 compares the ability of the formulation of the invention withthat of chemical treatments to effect whitefly scales.

FIG. 3 compares the ability of the formulation of the invention withthat of chemical treatments to effect whitefly eggs.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein is a process and method for preparing abiopesticide agent which can be applied to plants, in and/or on thesurface of soil for the control of plant pests, particularly insects, oron the surface of water to control pests such as mosquitos. Thepathogenic fungi which are useful for the purpose of this invention arepreferably the fungal species from the taxonomic classes as described byAinsworth et al. "The Fungi", vol. 4 a,b., Academic press (1973). Themajor Taxa which contain the entomogenic fungal species are from thefollowing subdivisions: Zygomycotina, Mastigomycotins, Ascomycotina,Basidiomycotina, and Deuteromycotina. These different subdivision can bepresented also by the different classes: Chytridiomycetes, Oomycetes,Zygomycetes, Plectomycets, Pyrenomcetes, Loculoascomycets, Teliomycetes,Coelomycetes, and Hyphomycetes.

Fungi from these classes which produce pathogenic spores that areinfective to pests can be used. For example, the following entomogenicfungi are considered to be the most suitable for pest control:Aspergillus, Aschersonia, Massospora, Beauveria, Metarrhizium,Verticillium, Paecilomyces, Hirsutella, Nomuraea, Hymenostilbe,Cordyceps, Coelomomyces, Lagenidium, Leptolegnia, Conidiobolus,Zoophthora, Culicinomyces, and Tolypocladium. Numerous strains of theseentomogenic fungi exhibit pathogenic activity against plant pests,mosquitos, and other animal pests. Most preferred are, the entomogenicfungi Paecilomyces, particularly the species Paecilomyces fumosoroseousATCC 20874. This particular strain is well suited for use as abiopesticide according to this invention.

FERMENTATION

The fungal biopesticides of the invention are prepared for delivery bygrowing selected fungal strain or strains, particularly the preferredPaecilomyces fumosoroseous strain ATCC 20874, in submerged culture.Although this description refers to the preparation of a single fungus,it will be appreciated that a mixture of genera or species may bedesirable in some applications. An inoculum of the preferred fungus orfungi may be prepared by a standard surface culture method in which thepreferred strain is grown on an agar slant, and the agar content is usedto inoculate shake flasks containing agar nutrient under standardconditions. After 48 hours of incubation the shake flask contains mainlyblastospores. This inoculum is used to inoculate fermenters usingstandard methods. It is within the scope of the invention to use anyconventional inoculation method.

The fermentation is conducted in such a way that the biomass in thefermenter substantially or predominately comprises filamentous mycelium,at least about 80% mycelium, preferably at least about 90% mycelium, andmost preferably at least about 95% mycelium. This can be achieved bysupplying an excess of complex carbon and complex nitrogen sources. Thecomplex carbon source can be naturally occurring mixtures such asmolasses, and the complex nitrogen source can be corn steep liquorand/or cotton seed flour. Other known complex carbon and complexnitrogen sources can also be used. Supply of pure sugars such assucrose, dextrose, glucose or other sugar sources will result in theformation of mainly blastospores and are therefore not preferred. Thecomposition of the nutrient media can be varied over a wide range.However, the preferred nutrient solution contains about 4 to 8 wt %complex carbon source and about 0.5 to 5 wt % complex nitrogen source.The production of mycelium can also be intensified by the constantsupply (fed batch addition) of molasses during the fermentation process.The fermentation can be carried out in batch, continuous or in fed batchmode.

In order to increase the yield of filamentous mycelium in the fermenter,it may be desirable to add stimulant nutrients to the cultivation media.Such stimulant nutrients include liquid fat, oils, surfactants, andpolyacids such as linoleic acid, silicon oils, water emulsion, etc.

A fed batch fermentation process can be advantageous to use, especiallywhen the pH of the fermentation is shifting toward acid conditions. Byadding molasses or other suitable complex carbon sources, it is possibleto maintain constant conditions in the fermentation without using largeamounts of base for pH adjustment. Fed batch addition of molasses, forexample, can be used as buffer control for the fermentation. Preferably,the addition Of molasses during fermentation is at a rate of about 1%molasses per 24 hours. Preferably, molasses addition begins after about48 hours or when the pH shifts toward acidic conditions (below about pH5.0).

The fermentation should be carried out in a manner such that agitationand aeration will be maximized. Any convenient means of aeration andagitation can be employed. If agitation is low, aggregation of myceliumoccurs. Therefore a minimum agitation rate of about 400 rpm to 600 rpmis preferred with aeration in the order of about 0.8 to 1 vvm. Thefermentation process preferably should be carried out in the temperaturerange of about 28°-30° C.

The fermentation should be carried out at an acidic pH sufficient topromote maximum formation of mycelium. Preferably, the pH is 4.0 to 6.0,most preferably about 5.0. Adjustment and control of the pH value can beachieved by the addition of an organic or inorganic base, preferablysodium hydroxide, ammonium hydroxide or any solution of triethyl amines.

In order to prevent developing undesirable amounts of foam duringfermentation, standard chemical defoaming agents can be added. Standardchemical defoaming agents include silicon oil, polypropylene or glycolcompounds, or other synthetic antifoams. The end of the cultivation canbe easily determined by the standard method of biomass determination(e.g., dry mass determination). Cultivation time will vary dependingupon such factors as, for example, the fungus or fungui used; thenutrients used; and the type of fermentation used. A 4 to 6 dayscultivation typically will be sufficient to yield 30-50 g/liter of drymycelium. This yield is sufficient for use in the formulation stage ofthis invention.

The separation of the filamentous mycelium biomass from the fermentationmedia can be accomplished by standard procedures such as filtration,centrifugation or other convenient means of separation. To avoidcontaminating the mycelium with undesirable microorganisms, harvestingthe mycelium under sterile conditions is recommended.

FORMULATION

In order to prepare the biopesticide formulation carrier of thisinvention, carrier materials are used which are capable of supportingfungal growth and promoting rapid sporulation. Suitable carriers includeinert filling compounds such as clay, bentonite, talcum, perlite,peatmoss, diatomaceous earth, kaolin, vermiculite, bran, dry milk, andminerals. Vermiculite is preferred because it results in a stablebiopesticide product and also because it lowers the density of theproduct, which allows the mycelium to conidiate faster. The vermiculite(or other carrier material) most preferably is pretreated to reduce thelevel of contaminating microorganisms. This preferably is by heating(i.e., at 100° C. or higher for at least up to about 1 hour) or may beby irradiation. Microbial decontamination may be also accomplishedchemically, provided that there is no retained chemical interferencewith fungal viability and growth. Returning to the preferred embodiment,the filamentous mycelium biomass is then added to the vermiculite orother carrier component by mixing.

Nutrients can be included in the mycelium formulation. Such nutrientsources may include carbon and nitrogen sources such as molasses, whey,milk powder, cotton seed flour, different autolyzed peptone, bran,wheat, malt extract or yeast extracts. It may be desired to addstabilization and/or protective agents to the formulation, such aspolyalcohols, glycerin or sugars. Antioxidants compounds such asascorbic acid or propyl gallate may be added, if desired.

The formulated mycelium mixture is then prilled by methods known to oneskilled in the art. The prilling process can be conducted by adding aprilling agent such as sodium alginate or potassium alginate to thebiomass/carrier mixture and by dropwise adding the mixture to acoagulant bath containing calcium chloride or calcium gluconate. Thesodium alginate concentration in the propagule mixture can vary fromabout 0.2 to about 3% depending on which formulation is used and degreeof propagulation needed. Calcium chloride or calcium gluconateconcentration for coagulating can vary from about 1 to about 15% w/v asneeded for suitable prill formation. Coagulation proceeds faster whenthe concentration of salts in the coagulation bath is increased. Theprill formed by this method can be dried immediately by using anyconvenient drying method, such as air drying or oven drying. However, afluidized bed process is preferred to obtain flowable prill withuniform, physical characteristics (shape, mechanical strength, size, anddensity).

In order to increase the conidiation potential of the prill, it may bedesired to add nutrients which will stimulate conidiation to the prill.Such stimulating material can contain natural food ingredients such asmolasses, peptone, cotton seed flour, glucose solution, etc. Theaddition of the nutrient can be before drying or in the drying process.Addition of nutrients before drying can be achieved by submerging theprill in a concentrated nutrient solution until diffusion is completeand then drying the treated prill. Addition of nutrients in the dryingprocess can be achieved by coating the prill during their movement inthe fluidized bed. Such coating procedures are well known. The prillshould be dried to a moisture or total volatiles content of about 2 toabout 12% (w/w). However, a total volatiles content of about 6-8% ispreferred. The moisture content can be easily determined according tostandard methods using moisture balances. To avoid contamination withundesirable microorganisms, the drying process should be done in sterileair, for example by using air filters.

Prill produced according to the invention can be stored under dryconditions at temperatures between about 4° C. to about 25° C. Topreserve the viability of the prill beyond 12 months, it is recommendedto store the prill in a vacuum or in an inert atmosphere such as underargon, nitrogen or other inert gases.

For purposes of this invention the term "prill" is used to mean astable, granulated particle or bead which does not produce a powder ordust and which has an average particle size of from about 0.2 mm toabout 5 mm in diameter.

REACTIVATION AND HARVEST

The formulated prill can optionally be reactivated prior to use. Thestable prill formulation can be reactivated prior to use to provide aconcentrate of highly active, fresh conidia spores. The formulated prillcontain optimum nutrients which stimulate sporulation and production ofa large number of spores in a short time upon activation. Each prill(less than 1 mm diameter) can produce as much as 10⁷ -10⁸ conidia sporesper prill and the conidia spores have high insecticidal effect oncontrolling pests. Reactivation is easily achieved by addition ofmoisture, preferably water, to the prill and allowing the growth ofmycelia and production of conidia spores. Optimal growth conditions areincubation at about 20°-28° C., preferably about 25° C. for less thanabout seven days, preferably about 3 to about 5 days. Reactivation cantake place in any suitable closed, sterile container, such as a petridish or tray. The prill preferably should be placed as a monolayer inthe container in order to achieve maximum surface area for thesporulation and germination process. The reactivated prill can be storedfor prolonged periods of time, i.e., up to about 1 year at about 10° C.to about 25° C., without losing conidiation activity or spore viability.

The conidia spores can be easily harvested from the reactivated prill bywashing with water or a non-toxic oil to provide aqueous or oilsuspensions of conidia spores. Preferably, the aqueous suspensioncontains a surfactant. Any suitable surfactant can be used, such as, forexample, a polyoxyethylene sorbitan monolaurate surfactant sold undertradenames Tween 80® or Tween 20® by Fisher Scientific of Fairlawn,N.J., in the range of 0.01-0.1%. Suitable oils include any oil which isnon-toxic such as, for example, cottonseed oil, vegetable oil, peanutoil, soybean oil, palm oil, sesame oil, jojoba oil, coconut oil, mineraloil or any other edible, non-toxic oil. Preferably, the oil iscottonseed oil. Conidia spores harvested by water can be usedimmediately or stored until application preferably, under coolconditions of about 4° C. Conidia spores harvested by oil can be storedfor up to a year, preferably about 6 months at room temperature (˜25°C.). In general, conidia spores in the amount of about 10⁷ -10⁸ sporeper prill may be obtained by cultivation of the prill in accordance withthe process of the invention. For purposes of the invention the term"non-toxic oil" is used to mean any oil which does not have any adverseor chronic toxiological properties to humans.

It is also within the scope of the invention to add nutrients to theaqueous suspension or non-aqueous oil suspension of conidia sporesbefore application to the targeted area of treatment. Such nutrients canenhance the germination of the conidia spores on the pests.Alternatively, it can be useful to pregerminate the conidia spores priorto application. Such pregermination can be easily established bystirring the harvested conidia spores in water for about 4-8 hours in acontainer or vessel or any other suitable means.

In another embodiment of the invention, the reactivated prill having theconidia spores attached thereto may be stored or packaged in awater-soluble polymeric container prior to harvesting the spores. Toharvest the conidia spores, the water-soluble polymeric container havingthe reactivated prill contained therein is dissolved in water and theresulting emulsion is filtered to provide an aqueous suspension ofconidia spores. Any polymeric material which is soluble in water, isinert to the fungal matter, and is capable of forming a container may beemployed in the invention. Preferably, the polymeric material is apolyvinyl alcohol. The polymeric materials may be formed into acontainer using conventional methodology known in the container orpackaging arts, such as, for example, by heat-sealing.

USE

The application of the dried prill, the reactivated sporulated prill,the harvested spores or the pregerminated spores prepared according tothe invention is dependent on the nature of the pest to be controlledand the particular field of use (i.e., agriculture, horticulture, forestor mosquito control). If the pest target is a plant pest, the prill, thereactivated prill or harvested conidia spores can be applied near theplant or on the plant. If the target is a soil pest, the dried prill,the reactivated prill or the conidia spores can be applied to or mixedwith the soil. If the target pests are mosquitos, the prill, thereactivated prill or the harvested conidia spores can be applied to thesurface of the water. An important economic use of biopesticides of thisinvention is in controlling insects or pests during shipment of plants.Application of the biopesticides on infested plants prior to shipmentwill result in obtaining plants free of insects.

Different entomogenic fungi can be used in the biopesticide prill of theinvention to control pests. Pests that can be controlled by thebiopesticides of this invention include arthropods and nematodes.Particularly preferred pests include insects such as mosquitoes andblackflies and pests belonging to the acarina and arachnid family. Thebiopesticides are effective against pests which are normally sensitiveand also those which are resistant to conventional pesticides. They areeffective against all or individual pest development stages. Thebiopesticides can be used effectively against pests from the following:Isopoda, Oniscus asellus, Armadillidium, Diplopoda, Chilopoda, Symphyla,Thysanura, Collembola, Orthopera, Dermaptera, Isoptera, Anoplura,Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera,Coleoptera, Hymenoptera, Diptera, Siphonaptera, Arachnidia, and Acarina.

Also, the biopesticides can be used against plant parasitic nematodesincluding Meloidogyne spp., Pratylenchus spp., Radopholus similes,Ditylanchus dipsaci, Heterodera spp., Xiphenema spp., Globodera spp. andHoplolaemus spp.

The principle target insect groups which are preferred for thebiopesticides of this invention are: Culicidae (mosquitoes) and otherDiptra, Aphidae (aphids), Dalphacidal (planthoppers), Cicadellidae(leafhoppers), Cercopidae (spittlebugs), Aleyodidae (white fly),Coccoidea (scales), Thysaoptera (thrips), Coleoptera (beetles), andLepidoptera (caterpillars).

The prill dosage will vary greatly depending on the application. Factorsto consider include the kind of prill formulation used (e.g.,vermiculite prills are more efficient in controlling mosquito larvaethan bran prills because of floating properties of the vermiculiteprills), the kind of pest, the state of crop infested with the pest, theprevailing weather conditions, and the kind of the agriculture area(e.g., agriculture, horticulture, forestry or other conditions). Ingeneral, for controlling plant insects, an application dosage range fromabout 10⁷ conidia spores/ml to about 10⁸ conidia spores/ml is preferred.Such dosage can be easily obtained based on the following ratio: onegram of reactivated prill yields a suspension of 10⁷ conidia spores/mlin one liter. For controlling soil insects, an application dosage ofabout 1-10 kg, preferably about 5 kg Of inactivated and/or reactivatedprill per hectare is preferred. The biopesticides can be applied by anyconvenient and conventional method including, broad cast spreading onthe soil or plant, or mixed with the soil.

The method of applying the biopesticides of the invention will varydepending on the particular biopesticide used and on the intended usethereof. The dried prill and the reactivated prill may be applied by anyconventional method known for applying dry granulated materials to thesoil, water or plants. For example, the dried prill or the reactivatedprill may be applied to the surface of soil and water by spraying orspreading using conventional apparatus. The dried prill and thereactivated prill may also be mixed into the soil for control ofsoilborne pests. Aqueous suspensions of the harvested conidia spores andthe pregerminated conidia spores may be applied directly to the soil orplants using conventional methodology such as spraying, pouring, etc.

The examples which follow are given for illustrative purposes and arenot meant to limit the invention described herein. The followingabbreviations have been used throughout in describing the invention.

CFU--Colony forming unit

CPU--Conidia spores per prill unit

°C.--degree(s) Centigrade

g--gram(s)

hr--hour(s)

l--liter(s)

μ--micro

%--percent

lb--pound(s)

ft² --square foot (feet)

M--molar

ml--milliliter

N--normal

PDA--peptone dextrose agar

RH--relative humidity

rpm--rotation per minute

w--weight

vvm--volume per volume per minute

AIS--Assessing Index Scale

GI--Growth Index

EXAMPLE 1 Production of Mycelium of Paecilomyces fumosoroseous in20-Liter Batch Fermenter

The fungus Paecilomyces fumosoroseous (ATCC 20874) was maintained on aslant agar containing 20 g/l malt, 20 g/l glucose, 1 g/l peptone, and 10g/l agar. The slant was stored at 4° C.

Slants were transferred under sterile conditions to a shake flask mediumof 30 g/l glucose, 20 g/l yeast extract, and 20 g/l corn steep liquor.The solution was adjusted to pH 6 before sterilization. Afterinoculation with the slant fungi, the shake flask containing the funguswas maintained at 30° C. for 24 hours on a round shaker at 300 rpm. Theshake flask product, mainly blastospores, was used to inoculate a20-liter fermenter containing 16 liters of production media composed of60-80 g/l molasses, 20 g/l cotton seed flour, and 20 g/l corn steepliquor. The fermentation pH was controlled to pH 5.3 by adding base (2MNaOH). Aeration was maintained at between 0.8-1.0 vvm, and agitation wasmaintained at 400-600 rpm. In order to avoid formation of foam, 1.5 mlof Macol® P-2000 antifoam agent (Mazer® chemicals) was added to thefermentation solution. The fermentation was completed after 96-100hours, and the filamentous mycelium was harvested by centrifugation. Theyield of filamentous mycelium obtained was 30 g/l (dry weight).

EXAMPLE 2 Preparation of Biopesticide Formulations

The mycelium of Paecilomyces fumosoroseous obtained in Example 1 wasused to prepare the formulations described in Table I. Briefly, thePaecilomyces mycelium (300 g at 25% moisture content) was blended andmixed with the described amounts of carriers. The carriers had beenpreviously autoclaved for 1 hour at 121° C. with 1 liter of water.Sodium alginate was added and each of the blended mixtures was broughtto a total volume of 3 liters and 1N NaOH was added to obtain theindicated pH. For prill formation of each mixture, a bath containing 5liters of a calcium chloride solution at concentration range of 13-27%(pH 6.35-7.00) was used. The blended mycelium/porous carrier mixture wasloaded onto a prilling column, and the mixture was added dropwise to thecoagulation bath to form alginate prill. The prill were submerged in thecoagulation bath for 1 hour or longer. The prill were easily removedfrom the bath by screening them through a metallic screen. The wetprill, which contain as much as 80-85% moisture, were loaded onto afluidized bed dryer and dried at an air temperature below 30° C. Theprill were dried to a water content of 6% to 10% w/w.

                  TABLE I                                                         ______________________________________                                                                         Alginate                                     Formulation                                                                            Porous Carrier                                                                              Amount    Amount pH                                    ______________________________________                                        A        Bran          300 g     60 g   6.0                                            Milk Powder    80 g                                                  B        Bentonite     500 g     35 g   8.5                                   C        Vermiculite   400 g     70 g   5.9                                   D        Peat Moss     350 g     35 g   8.5                                   E        Vermiculite   200 g     60 g   8.5                                            Bran          200 g                                                           Cotton Seed Flour                                                                           100 g                                                  F        Vermiculite   300 g     45 g   5.3                                            Bran          100 g                                                  G        Vermiculite   300 g     45 g   9.0                                            Bran          100 g                                                  ______________________________________                                    

EXAMPLE 3 Evaluation in vitro of Prill and Conidia Spore Viability

Batches of biopesticide prill prepared according to Example 2 wereevaluated and assessed for viability, germination and conidiation afterstorage.

Prill Mycelium Viability

Thirty-six prill were rehydrated by submerging in water for 30 minutes.The reactivated prill were placed on the surface of an agar plate orempty plastic well and incubated for 48 hours at room temperature. Thepercent viability of the prill was evaluated by observing and trackingthe mycelium developed on the surface of the prill. Table II representspercent prill viability of different batches which were stored atdifferent temperatures for up to 12 months.

                  TABLE II                                                        ______________________________________                                                                 Storage                                                         Storage       Time     Prill                                       Formulation                                                                              Temperature   (months) Viability                                   ______________________________________                                        D          4° C.  6        100%                                        D          25° C. 6         99%                                        A          4° C.  6        100%                                        A          25° C. 6         99%                                        A          4° C.  5         98%                                        A          25° C. 9         95%                                        A          25° C. 12        95%                                        A          4° C.  12        95%                                        B          4° C.  11       100%                                        C          4° C.  12       100%                                        F          4° C.  6        100%                                        G          4° C.  6        100%                                        ______________________________________                                    

Conidiation of Mycelium Prills In Vitro Test

Conidiation of the different prill formulations which sporulated on agarplates or empty plastic wells for 48 hours as above were examined bytransferring three sporulated prill to a sterile screw cap tubecontaining 10 ml of a 0.01% Tween™ 80 surfactant solution. The tube wasshaken vigorously for 20 seconds and the conidia spores were countedusing a hemacytometer. Table III represents spore counts for prill whichwere stored at different temperatures for as up to 11 months. Allconidia spores germinated to 100%.

                  TABLE III                                                       ______________________________________                                        Conidiation at Different Temperatures:                                                 Storage    Storage Time                                                                             Conidiation                                    Formulation                                                                            Temperature                                                                              (months)   (Spore per Prill)                              ______________________________________                                        A         4° C.                                                                            10         1.95 × 10.sup.7                          A        25° C.                                                                            10         1.07 × 10.sup.7                          A        25° C.                                                                            11         1.33 × 10.sup.7                          C        25° C.                                                                             6          5.0 × 10.sup.7                          F        25° C.                                                                             6         1.87 × 10.sup.7                          ______________________________________                                    

Conidia Spore Germination In Vitro Test

The conidia spores which were produced in the preceding section by thesporulated prill were collected and a 1×10⁵ spore per ml solution wasprepared. Sterile sabourads dextrose broth (100 μl) was added to 2 rowsof a 96 well plate (Corning 25860 polystyrene). The spore suspension(100 μl) was added to a total of 16 wells of each plate. The spores wereincubated at 28° C. for 24 hours and were examined for germination(forming germ tube). All prill formulations tested produced 100%germination of the conidia spores under the various storage conditionsand times.

EXAMPLE 4 Efficacy Study

The efficacy of the bran/milk biopesticide prill formulations preparedin Example 2 was evaluated in a greenhouse for control of whiteflies.Each test was conducted by infesting a blue salvia plant withwhiteflies. The insects were allowed to oviposit for 24 hours, then alladults were removed. The salvia were grown under greenhouse conditionsallowing the immature whitefies to develop. Several early to mid-forthinstar scales were removed and incubated in 100% relative humidity todemonstrate that no viable Paecilomyces fumosoroseous fungi were presentin the greenhouse. The infested plants were then subjected to thefollowing treatments:

Treatment A--1 g of prill per plant was scattered directly on themoistened surface of the soil.

Treatment B--1 g of prill per plant was soaked for 1 hour in deionizedwater. The prill were placed in a petri dish atop 1 piece of moistenedWhatman™ 5-filter paper and incubated for 24 hours. The prills were thenscattered directly on the soil surface.

Treatment C--10 prill were incubated on a PDA agar plate for 7 days at25° C. under a 12-hour photo period. The plate was scraped with asterile instrument. Five ml of this solution were pipetted into 500 mlof a 2% sucrose solution. Plants were dipped in this solution.

Treatment D--Plants were dipped in 500 ml of deionized water (no prilladded).

Eight plants were used per treatment. Living, dead, and infected scaleswere counted on 3 leaves per plant weekly. Each week, 24 instar scaleswere removed from leaves in each treatment and incubated in 100% RH tomeasure mortality. Table IV represents the mortality and efficacyresults.

                  TABLE IV                                                        ______________________________________                                                    Percent of Infected Whitefly                                      Treatment Type                                                                              Day 3      Day 7                                                ______________________________________                                        Treatment A   60%        94%                                                  Treatment B   48%        58%                                                  Treatment C   40%        60%                                                  Treatment D   18%        18%                                                  ______________________________________                                    

EXAMPLE 5 In Vivo Bio Assay (Reactivation of Prill)

A standard in vivo bio assay to assess the viability and infectivity ofthe conidia spores produced by the prills has been developed. Theprocedure consists of harvesting conidia spores from activated prills bymeans of soaking the prills in 0.05% Tween™ 80 solution and thendiluting the conidia spores harvested to obtain a concentration of1.0×10⁷ conidia spores per 1 ml which was used to assess efficacy.

Germination (Viability) Assays

The conidia spore suspension was spread over a sterile microscope slidecoated with a thin layer of water agar (2.0% Difco agar) by means of asterile inoculating loop. Slides were then placed into wet chambers(petri dishes with filter paper) and 0.2 ml of sterile water was addedto the filter paper. Conidia spores were incubated for 16 hours in anincubator (25° C.) and assessed for germination under a lightmicroscope. All conidia spores with a germ-tube(s) of any size werecounted as being germinated.

Infectivity Assay

The sweet potato whitefly B. tabaci was the host insect used formeasuring infectivity. Early 4th instar nymphs of B. tabaci(un-synchronized population reared on Hibiscus sp) were collected fromleaves with a flattened needle and placed on the surface of a sterilemicroscope slide. Conidia spore suspension which was prepared asdescribed above was used by placing a drop with known concentration onthe surface of a sterile microscope slide (20 drops per slide, 10 ineach of two rows running the length of each slide). Previously collectednymphs of B. tabaci were placed on each droplet and into a wet chamberand 0.2 ml of sterile water were added to the filter paper. Samples werekept in an incubator (25° C.) for a period of 7 days. Two chambers(total of 40 nymphs) were used for each sample tested. As a control,slides with nymphs only were prepared by the same manner as previouslydescribed but without conidia spores added to the Tween 80 solution. Toevaluate the growth of the fungus without nutrient source, slides withdrops of conidial suspension only (without nymphs) were also prepared.

Each nymph or drop of conidia spore was observed under a lightmicroscope (100×magnification) and rated according to the followingindex (Assessing Index Scale="AIS"):

0.0--No changes (conidia spores do not germinate, no visible changesanywhere in the drop area)

0.5--Germination of conidia spores (beginning of germination, conidiaspores with one or two germ-tubes are present anywhere in the drop areaor unbranched hyphae are present)

1.0--Beginning of mycelial growth (anywhere in the drop area presence ofbranched hyphae is visible, no growth of fungus on host is noted)

1.5--First occurrence of the fungus hyphae on the host (anywhere on thehost surface hyphae of fungus are present)

2.0--the surface or alongside of host body overgrowth by Regular growthof mycelium on the host (on most of mycelium is visible)

2.5--First occurrence of newly formed conidia spores (anywhere on thehost surface or on a mycelium alongside the host body firstconidiophores and newly formed conidia spores in chains are present)

3.0--Regular sporulation (fully sporulated mycelium covers most of thesurface of infected host or takes place alongside of host)

All samples were rated on days 1,3,5, and 7. The results were expressedas a daily average of "AIS" from each sample (40 nymphs). Control nymphswere assessed with the developmental stage of the nymph and presence ofany infection being recorded. Control drops (no nymphs of B. tabaciplaced into the drop) were observed and the developmental phase offungus noted (germination of conidia spores, mycelial growth, orsporulation).

Basic physical characteristics of different types of alginate prill usedin the experiment are presented in the following table.

                  TABLE V                                                         ______________________________________                                                  Avg Wt of 1 Avg Amt of Shape of                                     Formulation                                                                             prill (g)   prills per 1 g                                                                           prills                                       ______________________________________                                        G         0.0027      370        homogenous                                   D         0.0025      400        homogenous                                   C         0.0035      286        diverse                                      E         0.0037      270        homogenous                                   E         0.0030      333        homogenous                                   E         0.0017      590        homogenous                                   E         0.0014      714        homogenous                                   ______________________________________                                         *The E formulations differ by date of manufacture.                       

Visual and physical weight changes which occur during activation areshown in Table VI.

                                      TABLE VI                                    __________________________________________________________________________    Initial  Day 1   Day 2     Day 7                                              Formu-                                                                             weight                                                                            wt  change                                                                            wt  change                                                                              wt  change                                         lation                                                                             (g) (g) (%) (g) (%)   (g) (%)                                            __________________________________________________________________________    F    0.0027                                                                            0.0039                                                                            +44.4                                                                             0.0042                                                                            +55.6 0.0046                                                                            +70.4                                          G    0.0025                                                                            0.0040                                                                            +60.0                                                                             0.0044                                                                            +76.0 0.0050                                                                            +100.0                                         D    0.0035                                                                            0.0048                                                                            +37.1                                                                             0.0056                                                                            +60.0 0.0065                                                                            +85.7                                          C    0.0037                                                                            0.0053                                                                            +43.2                                                                             0.0055                                                                            +48.6 0.0046                                                                            +24.3                                          E    0.0030                                                                            0.0047                                                                            +56.7                                                                             0.0050                                                                            +66.7 0.0063                                                                            +110.0                                         E    0.0017                                                                            0.0033                                                                            +94.1                                                                             0.0035                                                                            +105.9                                                                              0.0032                                                                            +88.2                                          E    0.0014                                                                            0.0027                                                                            +92.9                                                                             0.0029                                                                            +107.1                                                                              0.0026                                                                            +85.7                                          __________________________________________________________________________

Table VII Compares different types of alginate pellets with incorporatedmycelium of entomogenous fungus Paecilomyces fumosoroseus isolate PFR97--an average growth index during activation of prills in laboratoryconditions (counted as an average GI from 20 prills*).

                  TABLE VII                                                       ______________________________________                                        Formu-                                                                        lation  GI - Day 1                                                                              GI - Day 3                                                                              GI - Day 5                                                                            GI - Day 7                                ______________________________________                                        F       1.8       3.4       4.6     5.0                                       G       1.1       3.3       4.4     5.0                                       D       1.4       3.6       4.1     5.0                                       C       0.8       1.8       3.8     5.0                                       E       2.0       3.6       4.4     5.0                                       E       1.8       3.0       4.2     5.0                                       E       1.7       3.2       4.4     5.0                                       ______________________________________                                         *Growth index scale:                                                          0 -- no visual changes on prills                                              1 -- swelling phase (change of size, shape and color, no growth of fungus     noted)                                                                        2 -- first hyphae growth on surface of prills                                 3 -- regular overgrowth, mycelium covers most of the prill's surface          4 -- beginning of sporulation (first conidiophores and conidial chains)       5 -- full sporulation (most of the prill's surface covered with sporulate     mycelium)                                                                

Production of conidia spores and germination of Formulation E over 7 dayperiod after activation is described in Table VIII.

                  TABLE VIII                                                      ______________________________________                                                         Germination Test                                                                              Shape of                                     Time of                                                                              An Average Amount of      Germinated                                   Activa-                                                                              Conidia Spores per 1                                                                          % Germin- Conidia                                      tion   Prill           ation     Spores                                       ______________________________________                                        Day 2  1.42 × 10.sup.6                                                                         93.7      2 germ - tubes                               Day 3  1.29 × 10.sup.7                                                                         98.8      2 germ - tubes                               Day 4  1.50 × 10.sup.7                                                                         100       2 germ - tubes                               Day 5  2.97 × 10.sup.7                                                                         100       2 germ - tubes                               Day 7  3.31 × 10.sup.7                                                                         100       2 germ - tubes                                Day 10                                                                              3.37 × 10.sup.7                                                                         99.2      2 germ - tubes                               ______________________________________                                    

Comparison of different types of prills for germination is shown inTable IX.

                  TABLE IX                                                        ______________________________________                                                                 Dominant Shape of                                                             Germinating Conidia                                  Formulation                                                                              % Germination Spores                                               ______________________________________                                        F          99.8          2 long germ-tubes (*)                                G          99.2          1 long germ-tube                                     D          93.3          2 short germ-tubes                                   C          81.6          1 short germ-tube                                    E          99.6          2 long germ-tubes (*)                                E          99.7          2 long germ-tubes (*)                                E          99.0          2 long germ-tubes (*)                                ______________________________________                                         (*)  first secondary branches presents                                   

Infectivity of the conidia spores produced from Formulation E afterdifferent periods of activation is described in Table X.

                  TABLE X                                                         ______________________________________                                        Period of                                                                              Average   Average   Average Average                                  Prills   "AIS"     "AIS"     "AIS"   "AIS"                                    Activation                                                                             Day 1     Day 3     Day 5   Day 7                                    ______________________________________                                        2 days   0.75      0.90      1.20    1.85                                     3 days   0.80      1.35      1.75    2.15                                     4 days   0.85      2.15      3.00    3.00                                     5 days   0.90      2.30      3.00    3.00                                     7 days   0.80      2.15      3.00    3.00                                     10 days  0.65      1.90      2.70    3.00                                     ______________________________________                                         *Assessing Index Scale  "AIS                                                  0.0 No changes (conidia spores do not germinate, no any visible changes       anywhere in a drop area)                                                      0.5 Germination of conidia spores (beginning of germination, conidia          spores with one or two germtubes are present anywhere in a drop area or       unbranched hyphae are present)                                                1.0 Beginning of mycelial growth (anywhere in a drop area presence of         branched hyphae is visible, no growth of fungus on host is noted)             1.5 First occurrence of the fungus hyphae on the host (anywhere on the        host surface a hyphae of fungus are present)                                  2.0 Regular growth of mycelium on the host (on most of the surface or         alongside of host body overgrowth by mycelium is visible)                     2.5 First occurrence of newly formed conidia spores (anywhere on the host     surface or on a mycelium alongside the host body first conidiophores and      newly formed conidia spores in chains are present)                            3.0 Regular sporulation (fully sporulated mycelium covers most of the         surface of infected host or takes place alongside of host)               

Comparison of different type of prill for infectivity is described inTable XI.

                  TABLE XI                                                        ______________________________________                                        Formu-    "AIS"-  "AIS"-      "AIS"-                                                                              "AIS"-                                    lation    Day 1   Day 3       Day 5 Day 7                                     ______________________________________                                        F         0.95    2.30        3.00  3.00                                      G         0.85    2.00        2.60  3.00                                      D         0.70    1.90        2.30  2.80                                      C         0.45    1.60        2.20  2.70                                      E         0.80    2.15        2.55  3.00                                      E         0.85    2.35        3.00  3.00                                      E         0.90    2.40        3.00  3.00                                      ______________________________________                                    

EXAMPLE 6

In order to compare the germination and infectivity of the conidiaspores produced by the prill to standard conventional methods of growingconidia spores on different substrate, the following experiment wasconducted. Three (3) samples of conidia spores were obtained from 3different sources:

1) from prills (formulation E),

2) from agar petri dishes (PDA--Potato Dextrose Agar Difco), and

3) from whitefly nymphs infected with PFR.

In all experiments, conidia spores of strain PFR ATCC 20874 were used.

The conidia spores from prills were harvested as previously described.The PDA-conidia spores were obtained from surface cultures on a solidartificial media. PDA plates were inoculated with 1 ml of conidialsuspension (1.0×10⁷ per ml), spread over the entire surface of the plateand kept an incubator (25° C.). When harvested on day 20, conidia sporesfrom the surface of the culture were washed with sterile 0.05% Tween 80surfactant and this suspension was diluted to a concentration of 1.0×10⁷conidia spores per 1 ml. The total amount of conidia spores produced ona surface cultures (PDA plates) was stated by the similar procedure asfrom activated prills. For the same experiment, conidia spores harvestedfrom infected hosts (B. tabaci, early 4^(th) instar nymphs) were used.The same method as for standard bioassay (described below) was used toobtain infected nymphs. Infected nymphs on which the fungus wassporulating were collected and placed into plastic ampules and soakedinto 1 ml of sterile 0.05% Tween 80 solution. The suspension of conidiaspores obtained in this manner was diluted to a concentration of 1.0×10⁷conidia spores per 1 ml. The total length (from tip of germ tube to thetip of the opposite one, if present) of at least 250 conidia spore wasmeasured using an ocular micrometer. The following table presents theviability and virulency of conidia spores obtained from PFR strain ATCC20874 which was obtained from different nutrient sources.

As can be seen from Table XII, the conidia spores obtained from theprill have a higher infectivity than those produced on the surface ofthe PDA culture. The time to obtain conidia spores on the PDA culture istwice as long as obtaining conidia spores from prills.

                  TABLE XII                                                       ______________________________________                                                       Dominant                                                                      Shape and                                                              %      Length of                                                              Ger-   Germinat-                                                      Nutrient                                                                              min-   ing Conidia                                                                             "AIS" "AIS" "AIS" "AIS"                              Source  ation  Spores    Day 1 Day 3 Day 5 7 day                              ______________________________________                                        Formula-                                                                              99.3   2 germ-   0.90  2.10  3.00  3.00                               ion E          tubes (4.4-                                                                   5.8 um                                                         PDA     93.8   1 germ-   0.75  1.85  2.60  3.00                                              tube (3.0-                                                                    3.8 um)                                                        B. tabaci                                                                             99.8   2 germ-   0.95  2.20  2.90  3.00                               E4 nymphs      tubes (8.2-                                                                   11.2 um)                                                       ______________________________________                                    

EXAMPLE 7

In order to evaluate the ability of PFR prill to sporulate and germinatein soil, the following experiment was conducted. Eight 250 ml Erlenmyerglass flasks were filled with 15 g of Redi lite soil mix (Terra-Lite®).The flask and the soil were autoclaved for 20 minutes at 121° C. Afterthe soil was cooled to room temperature, 20 vermiculite prill (asprepared and described in Example 2) were added to the soil mixture inthe flask. The soil and the prill were shaken well, and 50 ml of steriledeionized water was added to enhance humidity. The soil prill mixturewas incubated at 25° and 30° C. After a month the number of spores inthe soil were evaluated. The spore count was determined by the followingmethod:

CFU Determination

1. Aseptically mixed thoroughly the flask contents using a sterilespoonula.

2. Carefully removed 1 g of wet soil mixture.

3. Made 1:10 serial dilutions using 50 mM phosphate buffer pH 7.0.

4. Plated the sample by adding 100 μl of sample onto a Rose Bengal plus100 mg chlorampenicol plate and make a spread plate. Prepare triplicateplates.

5. Incubated the plates at 28° C.

6. After 5 to 6 days counted the fungal colonies present.

Table XIII represents the results of this study.

                  TABLE XIII                                                      ______________________________________                                                    CFU/g after 30 Days                                                           Incubation Temperature                                            Flasks        25° C.                                                                           30° C.                                         ______________________________________                                        Flask 1,2     2.48 × 10.sup.6                                                                   2.2 × 10.sup.6                                  Flask 3,4     2.0 × 10.sup.5                                                                    2.6 × 10.sup.6                                  Flask 5       2.6 × 10.sup.6                                                                    --                                                    ______________________________________                                    

EXAMPLE 8 Large Scale Protection of Hibiscus Against White Fly

The PFR conidia spores harvested from reactivated prills were tested toevaluate the ability to protect hibiscus sp. plants against white fly(Bemisia tabaci). The reactivated prill described in Example 5 were usedto protect hibiscus plants grown in commercial greenhouses in Apopka,Fla. Large quantities (100 g) of dried prill were activated in largeplastic boxes (450×250× 100 mm). The same procedures for evaluating andcultivating the prill as described in Example 5 were used. Briefly,after 7 days incubation at 25° C., the conidia spores produced werestored at 4° C. About 3-6 hours prior to application, 10 g of the sporeswere harvested from the reactivated prill into 2 liters of water plus0.05% Tween 20® surfactant (obtained by Fisher Scientific, Fairlawn,N.J.). The concentration of conidia spores in this concentrate wascounted using a Neubaer hemocytometer. Prior to application, the conidiaspore concentrate was diluted with water to a final concentration of1.0-1.5×10⁷ conidia spores/mi. Fifty liters of the final conidia sporesuspension were applied in one treatment to 800 hibiscus plants using abackpack sprayer (Birchmeier) with special attention to cover theunderside of the leaves. The biopesticide was applied weekly for aperiod of one month. As a control, Hibiscus plants were treated withdifferent chemical agents (see Table for chemical application). Plantswere checked once a week, and leaves were examined randomly from thechemical control and from the PFR treatment. FIGS. 2-4 show thecomparison of the treated plant with PFR to a control area which wastreated with chemicals. As can be seen from the figures, PFR worksbetter than the chemical treatment. It is important to note thatparasitized scales were found mainly in the chemical treatment and notat the PFR site. However, no parasites infected with PFR were found.Therefore, mortality of whitefly at the PFR treated area is mainlybecause of the fungus establishment.

EXAMPLE 9 Pregermination

Conidia spores harvested from reactivated prill described in Example 5(Formulation E) were evaluated to determine whether pregermination ofconidia spores or addition of stimulating nutrient to the conidia sporescan enhance the infectivity of the conidia spores. The conida wereapplied to plants infected with whiteflies.

The results shown in Table XIV indicate that pregerminated conidiaspores have the greatest effectiveness in controlling whitefly withinthe shortest time (12-24 hours).

                  TABLE XIV                                                       ______________________________________                                        Time at 100%                                                                  humidity                                                                      directly Percent of Dead Whitefly:                                            after    Formulation E                                                                             Formulation E                                                                             Formulation E                                Application*                                                                           in Water    with Nutrient                                                                             Pregerminated                                ______________________________________                                        12       64          72          84                                           24       56          77          82                                           48       47          91          89                                           72       68          94          90                                           ______________________________________                                         *Subsequently switched to 75% humidity                                   

EXAMPLE 10 Induced Resistance

The conidia spores of the invention were tested to determine theinfluence of the entomogenous fungus Paecilomyces fumosoroseus on thecolonization and induced resistance of host plants. A total of 60uninfested poinsettia plants of the same size and shape (an average of 6leaves/plant) grown in small pots were used. Adult sweetpotatowhiteflies were caught into plastic tubes and released onto the treatedplants. The plants were treated with Paecilomyces fumosoroseus PFR 97harvested from alginate pellets (Formulation E activated as described inExample 5). The conidial suspension used was a 0.05% Tween solutionadjusted to a titre of 1.0×10⁷ conidia spores per ml.

The treatments used were:

A--preventive treatment (plants were treated with conidial suspensionone week before exposure to sweetpotato whitefly adults. Before whiteflyadults were released, the treated plants were deposited into a nyloncage to prevent any undesirable infestation);

B--treatment before release of whitefly adults (plants were treated withconidial suspension and then exposed to the whitefly adults when thesurface of treated plants dried);

C--Control plants (plants were treated with 0.05% Tween solution andthen exposed to the whitefly adults when the surface of treated plantsdried).

All treated plants were placed in a greenhouse in a randomized squarefashion. Twelve plants from each of the variants (A-B-C) were placed ina randomized square and adults of whitefly were released at 5 points(center of the square and each of the corners in the second row).

The number of adults per plant was determined every 24 hrs for a periodof 1 week. When counted, the following data were noted:

a) alive adults (total amount per all plants in each group)

b) dead adults (total amount per all plants in each group)

c) infected adults (total amount per all plants in each group)

Treatment of plants with Paecilomyces fumosoroseus ATCC 20874 conidiaspores harvested from prills resulted in significantly poorerestablishment of pest populations when treated 1 week before exposurethan when treated just prior or not at all. Treatment just prior toexposure was better than not at all. These results shown in Table XVindicate that conidia spores of Paecilomyces fumosoroseus ATCC 20874harvested from prills illicit an immune response (induced resistance) bythe plant.

                  TABLE XV                                                        ______________________________________                                              Total number of                                                                            Variant A Variant B                                                                             Variant C                                Day   alive adults number/%  number/%                                                                              number/%                                 ______________________________________                                        1     1157         238/20.6  336/29.0                                                                              583/50.4                                 2     1146         170/14.8  379/33.1                                                                              597/52.1                                 3     967          130/13.4  298/30.8                                                                              539/55.8                                 4     697           71/10.2  186/26.7                                                                              440/63.1                                 5     447          34/7.6     93/20.8                                                                              320/71.6                                 6     315          19/6.0     70/22.2                                                                              226/71.8                                 7     270          23/8.5     63/23.3                                                                              184/68.2                                 14     68           10/14.7   22/32.4                                                                               36/52.9                                 ______________________________________                                    

EXAMPLE 11 Preparation of Aqueous Suspension of Conidia Spores UsingWater-Soluble Container of Reactivated Prill

Mycleium of the Fungus Paecilomyces fumosoroseus (ATCC 20874) wasproduced as described in Example 1. The mycelium obtained was formulatedwith a porous carrier and nutrient in the following ratio: 20% Bran; 20%vermiculate, 20% cotton Seen Floure (CSF) and 50% of mycelium.Formulated prill were obtained and dried as described in Example 2. Onehundred (100) grams of the prills were further activated as described inExample 5. Following a rich, full sporulation, after 7 days theactivated prill was dried by placing them in a box at room temperature(25° C.) and a relative humidity of 10-20%.

After 10 days of storage, 10 grams of the dried reactivated prill wereplaced in a polyvinyl alcohol release film (obtained from Chris CraftIndustrial Products, Inc.) 60×60 mm, and the film was heat-sealed toproduce a bag using an Electronic Impulse Autosealer Type 450 by TEWElectric Heating Equipment Co., LTD. The bag was then introduced into awater bath containing 100 ml of distilled water with 0.05% surfactant(Tween 80® obtained from Fisher Scientific of Fairlawn, N.J.). The bathwas agitated using a magnetic stirrer. After 1 hour of mixing thecontent of the bath was examined. The polymeric bag was completelydissolved to yield a suspension of conidia spores. The suspension wasfiltered to remove portions of the prill which were not dissolved. Thenumber of spores per ml of the suspension was 1.1×10⁸, as determined bycounting spores under a light microscope (100× magnification).

We claim:
 1. An improved stable, dried, prilled biopesticidalcomposition comprising an inert carrier which is capable of supportingfungal growth and promoting conidia sporulation, and an entomogenousfungal biomass at least about 80% of which is in the form of myceliumand is prepared by submerged fermentation of the fungus, Paecilomycesfumosoroeus isolate ATCC No.
 20874. 2. The biopesticidal composition ofclaim 1 wherein the fungus has been treated to produce conidial spores.3. The biopesticidal composition of claims 1 or 2 wherein at least about90% of the biomass is in the form of mycelium.
 4. The biopesticidalcomposition of claims 1 or 2 wherein the mycelium is filamentous inform.
 5. The biopesticidal composition of claims 1 or 2 which furthercomprises a nutrient.
 6. A process for preparing an improved fungalbiopesticide for control of insect and nematode infestationcomprising(a) fermenting a fungus Paecilomyces fumosoroseus isolate ATCCNo. 20874 in a culture medium by submerged fermentation to producebiomass such that at least 80% of the biomass is in the form ofmycelium; (b) harvesting the biomass; (c) mixing the biomass with acarrier; (d) forming prill from the biomass/carrier mixture; (e)optionally, drying the prill; (f) treating the prill to producepathogenic conidia spores; and (g) harvesting the conidia spores fromthe treated prill.
 7. An improved biopesticidal formulation forcontrolling insect and nematode pests comprising conidial spores of thefungus Paecilomyces fumosoroseus isolate ATCC 20874 wherein saidconidial spores have been prepared from a stable, dried, prilledbiopesticidal composition comprising an inert carrier which is capableof supporting and promoting growth of the fungus and a biomass of thefungus, at least 80% of which biomass is in the form of mycelium,wherein the fungal biomass is prepared by submerged fermentation.
 8. Thebiopesticidal formulation of claim 7 wherein the conidial spores havebeen pregerminated to produce germ tubes.
 9. The biopesticidalformulation of claim 7 which further comprises a mammalian non-toxicoil.
 10. The biopesticidal formulation of claim 9 in which the oil isselected from the group consisting of cottonseed oil, vegetable oil andmineral oil.
 11. The process of claim 6 which further comprises thefollowing step:(i) pregerminating the harvested conidia spores.
 12. Theprocess of claim 6 wherein at least about 90% of the biomass is in theform of mycelium.
 13. The process of claim 6 wherein the mycelium isfilamentous in form.
 14. The process of claim 6 wherein the conidiaspores are harvested by washing the treated prill with water or anon-aqueous, non-toxic oil.
 15. The process of claim 14 wherein thenon-toxic oil is selected from cottonseed oil, vegetable oil, peanutoil, palm oil, sesame oil, jojoba oil, coconut oil, soybean oil ormineral oil.
 16. A fungal biopesticide prepared by the process of claims11, 14, or
 6. 17. The biopesticide of claim 16 which is effective forthe control of insect and arachnid infestation.
 18. The biopesticide ofclaim 17 which is effective for the control of infestation bywhiteflies, mosquitos, aphids, planthoppers, leafhoppers, spittlebugs,mites, scales, thrips, beetles or caterpillars.
 19. The biopesticide ofclaim 16 which is effective for the control of nematode infestation orplant damage incurred by nematodes.
 20. A method for controlling insectand nematode infestation of a treatment area comprising applying apesticidally effective amount of the biopesticide of claim 16 to thearea.